Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)
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Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.
KeywordsAndrogenesis Callogenesis Chromosome doubling Cucurbits Embryogenesis Organogenesis
Thanks are due to all the whole staff of the Cell Biology Group for helping and training AA during his stay in the group. This work was supported by Grant AGL2017-88135-R to JMSS from Spanish Ministerio de Economía y Competitividad (MINECO) jointly funded by FEDER.
AA performed all the experimental work and analyzed the results. AZ, MRA and JMSS designed the experimental work and analyzed the results. JMSS wrote the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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